Three-dimensional Culture of Human Airway Epithelium in Matrigel for Evaluation of Human Rhinovirus C and Bocavirus Infections

Biomed Environ Sci. 2018 Feb;31(2):136-145. doi: 10.3967/bes2018.016.

Abstract

Objective: Newly identified human rhinovirus C (HRV-C) and human bocavirus (HBoV) cannot propagate in vitro in traditional cell culture models; thus obtaining knowledge about these viruses and developing related vaccines are difficult. Therefore, it is necessary to develop a novel platform for the propagation of these types of viruses.

Methods: A platform for culturing human airway epithelia in a three-dimensional (3D) pattern using Matrigel as scaffold was developed. The features of 3D culture were identified by immunochemical staining and transmission electron microscopy. Nucleic acid levels of HRV-C and HBoV in 3D cells at designated time points were quantitated by real-time polymerase chain reaction (PCR). Levels of cytokines, whose secretion was induced by the viruses, were measured by ELISA.

Results: Properties of bronchial-like tissues, such as the expression of biomarkers CK5, ZO-1, and PCK, and the development of cilium-like protuberances indicative of the human respiration tract, were observed in 3D-cultured human airway epithelial (HAE) cultures, but not in monolayer-cultured cells. Nucleic acid levels of HRV-C and HBoV and levels of virus-induced cytokines were also measured using the 3D culture system.

Conclusion: Our data provide a preliminary indication that the 3D culture model of primary epithelia using a Matrigel scaffold in vitro can be used to propagate HRV-C and HBoV.

Keywords: 3D cell culture; Human airway epithelium (HAE); Human bocavirus; Human rhinovirus C; Propagation.

MeSH terms

  • Collagen
  • Drug Combinations
  • Enterovirus / growth & development*
  • Enterovirus / isolation & purification
  • Enterovirus Infections / virology*
  • Enzyme-Linked Immunosorbent Assay
  • Epithelial Cells / virology
  • Human bocavirus / growth & development*
  • Human bocavirus / isolation & purification
  • Humans
  • Laminin
  • Parvoviridae Infections / virology*
  • Primary Cell Culture / methods*
  • Proteoglycans
  • Real-Time Polymerase Chain Reaction
  • Respiratory Mucosa / virology*
  • Virus Cultivation

Substances

  • Drug Combinations
  • Laminin
  • Proteoglycans
  • matrigel
  • Collagen

Supplementary concepts

  • Rhinovirus C